Human and mouse fibroblasts with normal p53 fail to enter mitosis when DNA synthesis is blocked by aphidicolin or hydroxyurea. Isogenic p53-null fibroblasts do enter mitosis with incompletely replicated DNA, revealing that p53 contributes to a checkpoint that ensures that mitosis does not occur until DNA synthesis is complete. When treated with N-(phosphonacetyl)-L-aspartate (PALA), which inhibits pyrimidine nucleotide synthesis, leading to synthesis of damaged DNA from highly unbalanced dNTP pools, p53-null cells enter mitosis after they have completed DNA replication, but cells with wild-type p53 do not, revealing that p53 also mediates a checkpoint that monitors the quality of newly replicated DNA.